Proposal Summary In the oral cavity, metabolic lactic acid production by bacteria and the associated change in pH are suspected to play a key role in the longevity and integrity of dental composite restorations. We propose gathering fundamental knowledge about the chemical microenvironment created by bacterial metabolites at the dental material interface to design next-generation dental composites. Our central hypothesis is that metal ion (Ca2+, Mg2+)-releasing composites can be engineered to influence bacterial metabolism and manipulate the chemical microenvironment to inhibit tooth demineralization. To quantify these bacterial chemical microenvironments, we will apply our newly developed unique electrochemical sensors (pH, lactate, H2O2, metal ions) to measure major bacterial metabolites such as lactate and H2O2 in real time. Aim 1: Determine the effects of metal ions on bacterial metabolism and the chemical microenvironment. We will determine the effects of metal ions on bacterial metabolism with dental plaque-derived microcosm biofilms such that the local pH is 5.5 or higher. To create a genetically amendable and reproducible biofilm model, we will extend our study to replicate local pH, lactate, and H2O2 concentrations with different ratios of three model organisms: Streptococcus mutans (lactate producing, pH lowering), Veillonella parvula (lactate consuming), and Streptococcus gordonii (H2O2 producing). We will use pH, lactate, and H2O2 microsensors as scanning electrochemical microscope (SECM) probes to determine the local rate of lactate and H2O2 production and the corresponding local pH change above the biofilms in real time in the presence of metal ions. Aim 2: Quantify the local pH above the bacterial biofilms grown on metal ion-releasing BAG composites. We will use SECM to measure pH at 20 m above the dental plaque microcosm and above three-species biofilm (Sm/Sg/Vp) grown on different metal ion-releasing composites (similar concentration ranges as in Aim 1). This will help us determine whether metal ions released from BAG composites can influence bacterial metabolism such that the local pH is >5.5. We will also use Ca2+- and Mg2+-sensing SECM probes to quantify the local concentration of metal ions released from BAG composites to determine the ion concentration to which bacteria will be exposed while growing on these composites. Aim 3: Measure pH and H2O2 at the biofilm?composite interface in real time. Innovative flexible wire sensors (pH, H2O2, metal ions) will be placed at the highly dynamic material?biofilm interface to monitor it and answer a crucial question: How do bacterial metabolites influence biomaterial integrity and how do metal ions released from biomaterials affect bacterial metabolites? The proposed research provides a significant step towards identifying next-generation ?smart? dental composites that can control biofilm composition to maintain a local pH of 5.5 or higher, thus inhibiting adjacent tooth demineralization and extending the lifespan of dental composite restorations.